Vaccine compositions

ABSTRACT

The present invention relates to the use of an excipient which is a compound of formula (I) or a physiologically acceptable salt or ester thereof: wherein: R 1  represents C 1-6 alkyl; R 2  represents hydrogen or C 1-6 alkyl; and R 3  represents C 1-6 alkyl, for increasing the immunogenicity of an influenza antigen, which use comprises (a) freezing, (b) heat-treating, and/or (c) freeze-drying an aqueous composition comprising the influenza antigen and the excipient.

FIELD OF THE INVENTION

The invention relates the use of specific dialkylglycine andtrialkylglycine excipients to increase the immunogenicity of influenzaantigens, as well as to methods for increasing the immunogenicity ofinfluenza antigens, to vaccine compositions obtainable using the method,and to the use of the vaccine compositions in vaccination of patients.

BACKGROUND TO THE INVENTION

Influenza virus is a member of the Orthomyxoviridae family. There arethree subtypes of influenza viruses designated A, B, and C that infecthumans.

Seasonal epidemics of influenza can spread around the world quickly andinflict a significant economic burden in terms of hospital and otherhealthcare costs and lost productivity. The World Health Organizationestimates that in annual influenza epidemics there are between three andfive million cases of severe illness and approximately 250,000 and500,000 deaths every year around the world.

An influenza pandemic occurs when a new influenza strain emerges in thepopulation with high pathogenicity and antigenic novelty. Globalpandemics can afflict between 20% and 40% of the world's population in asingle year. The pandemic of 1918-19, for example, affected 200 millionpeople, killing over 30 million worldwide. Although healthcare hasdramatically improved since that time, with vaccines and antiviraltherapies being developed, it is estimated that a pandemic today wouldresult in two to seven million deaths globally.

In the event that an influenza pandemic were to occur, one problem thatcould arise is that it might be difficult to manufacture sufficientquantities of the influenza antigens required for use in vaccines in therequired timescale. Another problem that could arise is that aninfluenza vaccines can take several weeks to confer immunity, which maynot be quick enough to prevent or reduce the spread of a highlyinfection strain.

There is therefore a need for influenza antigens with increasedimmunogenicity, which could be used in smaller quantities and/or conferimmunity more quickly than existing antigens.

WO 2011/121301, WO 2011/121306 and WO 2013/050780 are concerned with theuse of particular excipients, including dialkylglycines andtrialkylglycines such as dimethylglycine (DMG), for stabilising viralparticles and/or polypeptides. WO 2011/121305 is concerned with the useof similar excipients for stabilising aluminium salt adjuvant duringfreezing or drying. None of these references are concerned withincreasing the immunogenicity of influenza antigens.

Journal of Laboratory and Clinical Medicine (1990), 115(4), 481-6 byReap et al describes the immunomodulating capabilities ofdimethylglycine (DMG) in a rabbit model. The rabbits were force fed DMGprior to and after inoculation with an influenza antigen. Reap et aldoes not describe freezing, freeze-drying or heating the influenzaantigen in the presence of DMG prior to administration to the rabbits.

SUMMARY OF THE INVENTION

It is a surprising finding of the present invention that that theimmunogenicity of influenza antigens can be increased by freezing,freeze-drying or heating the influenza antigen in the presence ofdialkylglycines and trialkylglycines such as dimethylglycine (DMG). Theresulting modified influenza antigen has increased immunogenicity ascompared to the unmodified influenza antigen. The resulting modifiedinfluenza antigen also has increased immunogenicity as compared to aninfluenza antigen which has been mixed with thedialkylglycines/trialkylglycines but has not undergone freezing,freeze-drying or heating.

There are two significant advantages associated with the increasedimmunogenicity of the modified influenza antigen.

Firstly, the modified influenza antigen can illicit the same immuneresponse in a patient using a much lower dose than unmodified influenzaantigen. This “antigen sparing” property is highly advantageous,particularly in a pandemic situation where millions of patients need tobe vaccinated.

Secondly, the modified influenza antigen is capable of conferringimmunity onto the patient more quickly than the unmodified influenzaantigen. The faster onset of immunity is also highly advantageous,particularly in a pandemic situation where it is important to try tostop the spread of the pandemic.

Accordingly, the present invention provides use of an excipient which isa compound of formula (I) or a physiologically acceptable salt or esterthereof:

wherein:

R₁ represents hydrogen or C₁₋₆ alkyl;

R₂ represents C₁₋₆ alkyl; and

R₃ represents C₁₋₆ alkyl,

for increasing the immunogenicity of an influenza antigen, which usecomprises (a) freezing, (b) heat-treating, and/or (c) freeze-drying anaqueous composition comprising the influenza antigen and the excipient.

The invention further provides a method for increasing theimmunogenicity of an influenza antigen, said method comprising (a)freezing, (b) heat-treating, and/or (c) freeze-drying an aqueouscomposition comprising the influenza antigen and an excipient which is acompound of formula (I) or a physiologically acceptable salt or esterthereof:

wherein:

R₁ represents hydrogen or C₁₋₆ alkyl;

R₂ represents C₁₋₆ alkyl; and

R₃ represents C₁₋₆ alkyl.

The invention further provides a vaccine composition obtainable by saidmethod.

The invention further provides a said vaccine composition, for use inpreventing an influenza infection in a human or animal patient.

The invention further provides use of a said vaccine composition in themanufacture of a medicament for preventing an influenza infection in ahuman or animal patient.

The invention further provides a method of preventing an influenzainfection in a human or animal patient, the method comprisingadministering to said patient a said vaccine composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Haemagglutination Inhibition Assay (HIA) titre measuredat various time points for mice administered Compositions A to F at days1 and 24 in Example 2. Compositions A to C were not been treated withexcipient. Composition A contained a normal dose of antigen, whereasCompositions B and C contained 1/100 dose of antigen. Compositions D toF contained 1/100 dose of antigen, but were admixed with excipients andthen heat-treated. The HIA titre for Compositions D to F wassignificantly than that higher observed with control Compositions B andC.

FIG. 2 shows the Haemagglutination Inhibition Assay (HIA) titre measuredat various time points for mice administered Compositions G to N at days1 and 24 in Example 3. The mice were considered to have acquiredimmunity once the HIA titre was greater than 50. Immunity was acquiredmost quickly with the combination of excipient, adjuvant and heattreatment (Composition N).

FIG. 3 shows the Haemagglutination Inhibition Assay (HIA) titre measuredat various time points for mice administered Compositions O to R at days1 and 24 in Example 4. These results demonstrate that the effect oninfluenza antigen observed with polyethyleneimine in Examples 1 and 2and Composition Q or Example 3 are also observed with dimethylglycine inComposition R of Example 3.

DETAILED DESCRIPTION OF THE INVENTION Summary

The present invention relates to increasing the immunogenicity of aninfluenza antigen using excipients of formula (I) or physiologicallyacceptable salt or ester thereof, such as dimethylglycine.

The influenza antigen is typically admixed with the excipient to give anaqueous composition, and the aqueous composition is then subjected to atreatment, such as freezing, heating and/or freeze-drying, thatincreases the immunogenicity of the influenza antigen. Freezing, heatingand/or freeze-drying the influenza antigen in the presence of theexcipient increases the immunogenicity of the antigen as compared tothat observed if the influenza antigen is merely mixed with theexcipient without freezing, heating and/or freeze-drying.

The influenza antigen and excipient interact during the treatment,thereby to increase the immunogenicity of the influenza antigen, ascompared to the immunogenicity of the influenza antigen prior to thetreatment. The immunogenicity of the antigen is therefore typicallyincreased during the treatment step.

Typically, the treatment is freezing or heating or freeze-drying,preferably freezing or heating, more preferably freezing. Alternatively,a combination of treatments may be used, such as freezing followed byheating or freeze-drying followed by heating. In the latter case, thefreeze-dried composition would typically be reconstituted prior toheating.

The resulting composition can be can be thawed, reconstituted or cooledafter freezing, freeze-drying or heating respectively, and administeredas a vaccine composition to a patient.

Aqueous Composition

The aqueous composition comprises the excipient and the influenzaantigen. The aqueous composition is typically a suspension or solution.The aqueous composition be prepared by admixing the excipient with theinfluenza antigen in an aqueous solvent. Any suitable aqueoussolvent-system may be used. The aqueous solvent may be buffered water.The aqueous solvent is typically HEPES-buffered water, Tris-bufferedwater, phosphate-buffered water or pure water.

Optionally, one or more sugars is admixed with the aqueous solvent priorto admixture with the excipient and influenza antigen. Alternatively theone or more sugars can be admixed with aqueous solvent after theexcipient and influenza antigen.

Optionally, an adjuvant is admixed with the aqueous solvent prior toadmixture with the excipient and influenza antigen. Alternatively theadjuvant can be admixed with aqueous solvent after the excipient andinfluenza antigen.

Typically, if an adjuvant is present, one or more sugars will also bepresent, since the one or more sugars will generally stabilise theadjuvant, particularly during the treatment step.

Other components may also be present in the aqueous composition. Forexample, a compound of formula (II) may also be present:

wherein X represents —S(O)₂— and R_(a) and R_(b) independently representC₁₋₆ alkyl. A preferred compound of formula (II) ismethylsulfonylmethane (MSM) in which R_(a) and R_(b) both representmethyl. The combination of excipient and compound of formula (II) mayinteract together, thereby to further increase the immunogenicity of theinfluenza antigen during the treatment step.

The concentration of excipient in the aqueous composition is typicallyin the range of 0.001M or more, preferably in the range of 0.01M or moreand more preferably 0.1M or more, for example from 0.1M to 5.0M, orabout 0.5M.

If one or more sugar(s) is used, the concentration of sugar or totalconcentration of sugar in the aqueous composition is typically 1M orless, preferably 0.7M or less, for example 0.5M or less or 0.3M or less.The sugar concentration or the total concentration may be down to 0.1 mMor to 0.5 mM.

The particular concentration of each component that is employed willdepend on several factors including the nature of the influenza antigen;the excipient being used; whether one or more sugar is being used and ifso the identity of the sugar(s); whether or not an adjuvant is present;and the particular freezing, freeze-drying or heat treatment procedurethat is adopted.

The Influenza Antigen

An influenza antigen suitable for use in the invention includes anyimmunogenic component of an influenza (types A, B or C) vaccine.

The influenza antigen may be a whole inactivated influenza virus or alive attenuated influenza virus. The influenza antigen may be a surfaceprotein of the influenza (types A, B or C). In particular, the influenzaantigen may be a hemagglutinin (HA), neuraminidase (NA), nucleoprotein,M1, M2, NS1, NS2(NEP), PA, PB1, PB1-F2 and or PB2 protein, or animmunogenic derivative or fragment of any of these proteins. Theinfluenza antigen may be HA1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9,HA10, HA11, HA12, HA13, HA14, HA15 and/or HA16, any immunogenic fragmentor derivative thereof and any combination of the HA proteins, fragmentsor derivatives. The neuraminidase may be neuraminidase 1 (N1) orneuraminidase 2 (N2).

The Excipient

The excipient is a compound of formula (I) or physiologically acceptablesalt or ester thereof.

The physiologically acceptable salt is typically a salt with aphysiologically acceptable acid and thus includes those formed with aninorganic acid such as hydrochloric or sulphuric acid or an organic acidsuch as citric, tartaric, malic, maleic, mandelic, fumaric ormethanesulphonic acid. The hydrochloride salt is preferred.

The ester is typically a C₁₋₆ alkyl ester, preferably a C₁₋₄ alkylester. The ester may therefore be the methyl, ethyl, propyl, isopropyl,butyl, isobutyl or tert-butyl ester. The ethyl ester is preferred.

As used herein, a C₁₋₆ alkyl group is preferably a C₁₋₄ alkyl group.Preferred alkyl groups are selected from methyl, ethyl, propyl,isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl areparticularly preferred.

For the avoidance of doubt, the definitions of compounds of formula (I)also include compounds in which the carboxylate anion is protonated togive —COOH and the ammonium cation is associated with a pharmaceuticallyacceptable anion. Further, for the avoidance of doubt, the compoundsdefined above may be used in any enantiomeric form.

Typically, R₁ represents hydrogen or C₁₋₄ alkyl, preferably hydrogen orC₁₋₃ alkyl, more preferably hydrogen, ethyl or methyl, most preferablyhydrogen or methyl.

Typically, R₂ represents C₁₋₄ alkyl, preferably C₁₋₃ alkyl, morepreferably ethyl or methyl, most preferably methyl.

Typically, R₃ represents C₁₋₄ alkyl, preferably C₁₋₃ alkyl, morepreferably ethyl or methyl, most preferably methyl.

R₂ and R₃ may be the same or different, but are preferably the same.When R₁ represents C₁₋₆ alkyl, then R₁, R₂ and R₃ may be the same ordifferent, but are preferably the same.

In a preferred embodiment, R₁ represents hydrogen and R₂ and R₃ are asdefined above. Thus, it is particularly preferred that R₁ representshydrogen and R₂ and R₃ represent methyl, such that the compound offormula (I) is dimethylglycine.

In an alternative preferred embodiment, R₁ represents C₁₋₆ alkyl and R₂and R₃ are as defined above. Thus, it is particularly preferred that R₁to R₃ all represent methyl, such that the compound of formula (I) istrimethylglycine.

Alternatively, instead of being a compound of formula (I) or aphysiologically acceptable salt or ester thereof, the excipient may be apolymer, such as polyethyleneimine (PEI).

PEI is an aliphatic polyamine characterised by the repeating chemicalunits denoted as —(CH₂—CH₂—NH)—. Reference to PEI herein includes apolyethyleneimine homopolymer or copolymer. The polyethyleneiminecopolymer may be a random or block copolymer. For example, PEI mayconsist of a copolymer of polyethyleneimine and another polymer such aspolyethylene glycol (PEG). The polyethyleneimine may be linear orbranched.

Reference to PEI also includes derivatised forms of a polyethyleneimine.A polyethyleneimine contains nitrogen atoms at various positions.Nitrogen atoms are present in terminal amino groups, e.g. R—NH₂, and ininternal groups such as groups interrupting an alkyl or alkylene groupwithin the polymer structure, e.g. R—N(H)—R′, and at the intersection ofa polymer branch, e.g. R—N(—R′)—R″ wherein R, R′ and R″ may be alkylenegroups for example. Alkyl or aryl groups may be linked to the nitrogencentres in addition to or instead of hydrogen atoms. Such alkyl and arylgroups may be substituted or unsubstituted. An alkyl group would betypically a C₁-C₄ alkyl group, e.g. methyl, ethyl, propyl, isopropyl,butyl, sec.butyl or tert.butyl. The aryl group is typically phenyl.

The PEI may be a polyethyleneimine that has been covalently linked to avariety of other polymers such as polyethylene glycol. Other modifiedversions of PEI have been generated and some are available commercially:branched PEI 25 kDa, jetPEI®, LMW-PEI 5.4 kDa, Pseudodendrimeric PEI,PEI-SS-PEI, PEI-SS-PEG, PEI-g-PEG, PEG-co-PEI, PEG-g-PEI, PEI-co-Llactamide-co-succinamide, PEI-co-N-(2-hydroxyethyl-ethylene imine),PEI-co-N-(2-hydroxypropyl) methacrylamide, PEI-g-PCL-block-PEG,PEI-SS-PHMPA, PEI-g-dextran 10 000 and PEI-g-transferrin-PEG,Pluronic85®/Pluronic123®-g-PEI. The PEI may be permethylatedpolyethyleneimine or polyethyleneimine-ethanesulfonic acid.

PEI is available in a broad range of number-average molar masses (M_(n))for example between 300 Da and 800 kDa. Preferably, the number-averagemolar mass is between 300 and 2000 Da, between 500 and 1500 Da, between1000 and 1500 Da, between 10 and 100 kDa, between 20 and 100 kDa,between 30 and 100 kDa, between 40 and 100 kDa, between 50 and 100 kDa,between 60 and 100 kDa, between 50 and 70 kDa or between 55 and 65 kDa.A relatively high M_(n) PEI of approximately 60 kDa or a relatively lowM_(n) of 1200 Da is suitable.

Preferably, the weight-average molar mass (M_(w)) of PEI is between 500Da and 1000 kDa. Most preferably, the M_(w) of PEI is between 500 Da and2000 Da, between 1000 Da and 1500 Da, or between 1 and 1000 kDa, between100 and 1000 kDa, between 250 and 1000 kDa, between 500 and 1000 kDa,between 600 and 1000 kDa, between 750 and 1000 kDa, between 600 and 800kDa, between 700 and 800 kDa. A relatively high M_(w) of approximately750 kDa or a relatively low M_(w) of approximately 1300 Da is suitable.

The weight-average molar mass (M_(w)) and number-average molar mass(M_(n)) of PEI can be determined by methods well known to those skilledin the art. For example, M_(w) may be determined by light scattering,small angle neutron scattering (SANS), X-ray scattering or sedimentationvelocity. M_(n) may be determined for example by gel permeationchromatography, viscometry (Mark-Houwink equation) and colligativemethods such as vapour pressure osometry or end-group titration.

Various forms of PEI are available commercially (e.g. Sigma, Aldrich).For example, a branched, relatively high molecular weight form of PEIused herein with an M_(n) of approximately 60 kDa and a M_(w) ofapproximately 750 kDa is available commercially (Sigma P3143). This PEIcan be represented by the following formula:

A relatively low molecular weight form of PEI used herein is alsoavailable commercially (e.g. Aldrich 482595) which has a M_(w) of 1300Da and M_(n) of 1200 Da.

Sugars

One or more sugars is optionally present in the aqueous composition. Twoor more sugars may be present, for example two, three or four sugars. Itis preferred that one or two sugars is present, most preferably twosugars. The combination of excipient and sugar(s) may interact together,thereby to increase further the immunogenicity of the influenza antigenduring the treatment step. The sugar(s) also assist in stabilising theadjuvant when present, particularly aluminium salt adjuvants, during thetreatment step.

The sugar is typically a monosaccharide, a disaccharide, atrisaccharide, a tetrasaccharide, a sugar alcohol or anotheroligosaccharide.

Typically, the monosaccharide is glucose, fructose, arabinose,glyceraldehydes, galactose or mannose. Typically, the dissaccharide issucrose, trehalose, lactose, cellobiose, turanose, maltulose, melibiose,isomaltose, or maltose. Typically, the trisaccharide is raffinose,melezitose or umbelliferose. Typically, the tetrasaccharide isstachyose. Typically, the sugar alcohol is mannitol. Other examples ofoligosaccharides include the pentasaccharide verbascose.

Typically, the sugar is a non-reducing sugars, for example sucrose orraffinose.

When one sugar is present in the aqueous solution, the sugar ispreferably mannitol or sucrose, preferably mannitol.

When two sugars are present in the aqueous suspension, the sugars arepreferably sucrose and raffinose.

Adjuvant

An adjuvant is optionally present in the aqueous composition. Anysuitable adjuvant may be used, but aluminium salt adjuvants arepreferred. When an aluminium salt adjuvant is used, it is preferred thatone or more sugars is also present in the aqueous composition, tostabilise the adjuvant during the treatment step.

Typically, the aluminium salt aluminium hydroxide (Al(OH)₃), aluminiumphosphate (AlPO₄), aluminium hydrochloride, aluminium sulphate, ammoniumalum, potassium alum or aluminium silicate. Preferably, the aluminiumsalt adjuvant used is aluminium hydroxide or aluminium phosphate. Mostpreferably, the aluminium salt adjuvant is aluminium hydroxide(Al(OH)₃).

Typically, the aluminium salt adjuvant takes the form of a hydrated gelmade from an aluminium salt, the hydrated gel being a particulatesuspension in aqueous media. The preparation of aluminium-salt adjuvantsare well known to those skilled in the art. For example, aluminiumhydroxide and aluminium phosphate adjuvants are generally prepared byexposing aqueous solutions of aluminium ions (typically as sulfates orchlorides) to alkaline conditions in a well-defined and controlledchemical environment, as known to those skilled in the art. Such methodscan be used for example, to prepare an aluminium hydroxide or aluminiumphosphate hydrated gel.

Freezing

Freezing of the aqueous composition can be conducted by any suitablemethod. Freezing may thus be carried out by immersing in liquid nitrogenor liquid nitrogen vapour, placing in a freezer or using a dry ice andalcohol freezing bath.

Typically, the aqueous composition is frozen to −4° C. or below,preferably −10° C. or below, more preferably to −20° C. or below, morepreferably to −30° C. The aqueous composition is typically not frozenbelow −100° C. The aqueous composition may, for example, be frozen toabout −80° C.

The aqueous composition is typically kept frozen at the desiredtemperature for 30 minutes or more, preferably 1 hour or more, forexample from 2 to 24 hours.

The frozen aqueous composition is typically allowed to thaw by leavingat room temperature before use as a vaccine.

The freezing and thawing conditions can be suitably optimised viaroutine experimentation.

Freeze-Drying

Freeze-drying can be carried out according to standard procedures. Thereare three main stages: freezing, primary drying and secondary drying.Freezing is typically performed using a freeze-drying machine. In thisstep, it is important to cool the biological material below its eutecticpoint, the lowest temperature at which the solid and liquid phase of thematerial can coexist. This ensures that sublimation rather than meltingwill occur in the following steps. Alternatively, amorphous materials donot have a eutectic point, but do have a critical point, below which theproduct must be maintained to prevent melt-back or collapse duringprimary and secondary drying.

During primary drying the pressure is controlled by the application ofappropriate levels of vacuum whilst enough heat is supplied to enablethe water to sublimate. At least 50%, typically 60 to 70%, of the waterin the material is sublimated at this stage. Primary drying may be slowas too much heat could degrade or alter the structure of the biologicalmaterial. A cold condenser chamber and/or condenser plates providesurfaces on which the water vapour is trapped by resolidification.

In the secondary drying process, water of hydration is removed by thefurther application of heat. Typically, the pressure is also lowered toencourage further drying. After completion of the freeze-drying process,the vacuum can either be broken with an inert gas such as nitrogen priorto sealing or the material can be sealed under vacuum.

The freeze-dried composition is reconstituted as an aqueous composition,using for example water or an aqueous buffer, before use as a vaccine.

The freeze-drying conditions can be suitably optimised via routineexperimentation.

Heat Treating

Heating treating of the aqueous composition can be conducted by anysuitable method.

Typically, the aqueous composition is heated to greater than 30° C.,preferably greater than 40° C. More preferably, aqueous composition isheated to a temperature of 30° C. to 80° C., for example 35° C. to 60°C. or 40° C. to 50° C. A preferred temperature is about 45° C.

Once the aqueous composition has been heated to the desired temperature,is preferably maintained at that temperature for 30 minutes or more,preferably 1 hour or more, for example from 2 hours to 2 weeks or from 4hours to 24 hours.

A typical heat treatment involves heating to about 45° C. andmaintaining at that temperature for about 7 days.

The heat treated aqueous composition is typically allowed to return toroom temperature before use as a vaccine.

The heat treatment conditions can be suitably optimised via routineexperimentation.

Increase in Immunogenicity

The immunogenicity of the influenza antigen is the ability of thatantigen provoke an immune response in the body of a human or animal. Achange in immunogenicity can be measured by comparing the immunogenicityof an unmodified (control) influenza antigen with the immunogenicity ofan influenza antigen modified in accordance with the invention, using astandard assay for predicting the level of immune response, such as thehaemmagglutination inhibition assay.

The immunogenicity of an influenza antigen can be measure using anysuitable technique known to those skilled in the art. A preferredstandard technique is the haemmagglutination inhibition assay. Anexemplary protocol for this assay is set out in the Examples below.

The increased immunogenicity of the modified influenza antigens of theinventions means that the same immune response can be obtained with asmaller amount of modified antigen. Thus, if a patient is administered adose D of unmodified antigen achieves a level of immunogenicity, thentypically a dose of 0.5 D or less, preferably 0.1 D or less, morepreferably 0.01 D or less, will achieve the same level ofimmunogenicity. The immunogenicity is typically measure at least 10 daysafter administration to the patient, for example after 10 days, 15 daysor 20 days.

The increased immunogenicity of the modified influenza antigens of theinvention means that the onset of immunity occurs more quickly than withunmodified antigen. Thus, if a patient takes a time T to acquireimmunity following administration of a given dose of unmodified antigen,then it will typically take 0.75 T or less time, preferably 0.5 T orless time, most preferably 0.2 T or less time, for a patientadministered the same dose of modified antigen to acquire immunity.

Use of Vaccine Compositions

Following freezing, freeze-drying, or heating, the resulting compositioncan be can be thawed, reconstituted or cooled respectively, and thenused as a vaccine composition. The vaccine composition can be diluted asnecessary with, for example, phosphate-buffered saline or Water forInjection, prior to use.

Typically, the vaccine composition contains one or more differentinfluenza antigens, at least one of which has been prepared inaccordance with the invention. In an embodiment, the vaccinecompositions contains two, three or four different influenza antigens,preferably all of which have been prepared in accordance with theinvention.

The resulting vaccine composition can then be administered by, forexample, injection, to a human or animal patient in need of vaccination.Typically, the patient is a human.

Preferably, the patient is a human who is part of the “at-risk”population. Such patients include children, elderly patients and/orpatients suffering from lung diseases, diabetes, cancer, or kidney orheart problems. This patient group is particularly preferred fortreatment with the vaccine compositions of the invention, because therapid onset of immunity achieved with the present vaccines compared tostandard vaccines reduces the risk of the patients being infected in thetime period between vaccination and onset of immunity.

Preferably, the patient has previously suffered an adverse-reaction toan influenza vaccine. This patient group is particularly preferred fortreatment with the vaccine compositions of the invention, because theantigen-sparing effect means that it is necessary to deliver less of thevaccine composition to the patient and accordingly that the risk of anallergic or other adverse reaction to the vaccine is reduced.

The following Examples illustrate the invention.

Example 1—Preparation of Vaccine Compositions Materials

Dimethylglycine (DMG), sucrose, raffinose, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, sodiumchloride and sterile water were all used as obtained from a commercialsource (Aldrich). Alhydrogel™ was used as obtained from a commercialsource (Source Bioscience). Polyethyleneimine (PEI) was used as obtainedfrom a commercial source (Sigma catalogue number: P3143—solution 50% w/vin water; M_(n) 60,000). Whole inactivated influenza A/SolomonIslands/2006 H1N1 antigen was obtained from the National Institute forBiological Standards and Control (NIBSC).

Composition Preparation

150 ml of HEPES buffer was prepared as follows. 100 ml of sterile waterwas measured out in a cylinder. 2.5 g NaCl was weighed out on acalibration checked balance and dissolved in the sterile water using amagnetic stirrer and bar. 6 ml of 1M HEPES was added and stirred. Whenfully dissolved, a pH meter and sodium hydroxide were used to alter thepH to 7.9. The final volume was made up to 150 ml with sterile water.The final HEPES buffer mix was then filtered through a 0.2 μm filterunit in a biological safety cabinet.

After weighing the required excipient components set out in Tables 1 to3 below, they were placed into a 50 ml sterile flask. Approximately 6 mlof the HEPES buffer was then added to the flask and the contents weremixed by swirling. The flask and contents were then heated in a +60° C.water bath until dissolved. When dissolved, the volume of the mix wasmeasured and the final volume made up to 10 ml with more HEPES buffer.The excipient formulation was filtered through a 0.2 μm filter unit in abiological safety cabinet.

Vials of freeze-dried influenza virus antigen A/Solomon Islands/2006H1N1 were allowed to reach room temperature from −20° C. and workingstocks of the antigen in sterile water were prepared.

Glass screw capped vials (pre autoclaved) were be prepared as detailedin Tables 1 to 3 below. Each vial contain was ultimately to containtotal volume of 1 ml. Where required, 500 μl of Alhydrogel™ stock (at aconcentration of 2% w/v, to give a final concentration of 1% w/v) waspipetted into each vial. The influenza virus antigen stock was thenpipetted into each vial so as to give a final antigen concentration of 2or 0.2 μg of antigen in each vial. The vials were capped and mixed byvortexing briefly.

The vials were then optionally treated with one of Treatments A to Cdescribed below and as set out in Table 1 to 3:

-   -   Treatment 1—the vials were stored at 45° C. for 7 days and then        allowed to return to room temperature.    -   Treatment 2—the vials were frozen to −80° C. and then allowed to        thaw by standing at room temperature.    -   Treatment 3—the vials were freeze-dried in a commercial        freeze-drier, by freezing to −80° C. and drying for 16 hours        under vacuum. The vials were then stored at 45° C. for 7 days        and then reconstituted back to a total volume of 1 ml and        allowed to return to room temperature.

TABLE 1 Influenza Composition antigen Adjuvant Excipient Treatment A 2μg No None None B 0.02 μg No None None C 0.02 μg No None 1 D 0.02 μgAlhydrogel PEI - 83 μM 1 Sucrose - 73 mM Raffinose - 21 mM E 0.02 μgNone PEI - 83 μM 2 Sucrose - 73 mM Raffinose - 21 mM F 0.02 μgAlhydrogel PEI - 83 μM 2 Sucrose - 73 mM Raffinose - 21 mM

TABLE 2 Influenza Composition antigen Adjuvant Excipient Treatment G 2μg None None None H 2 μg None None 2 I 2 μg None PEI - 83 μM NoneSucrose - 73 mM Raffinose - 21 mM J 2 μg None PEI - 83 μM 2 Sucrose - 73mM Raffinose - 21 mM K 2 μg Alhydrogel None None L 2 μg Alhydrogel None2 M 2 μg Alhydrogel PEI - 83 μM None Sucrose - 73 mM Raffinose - 21 mM N2 μg Alhydrogel PEI - 83 μM 2 Sucrose - 73 mM Raffinose - 21 mM

TABLE 3 Influenza Composition antigen Adjuvant Excipient Treatment O 2μg None None None P 2 μg None None 3 Q 2 μg None PEI 3 Sucrose - 300 mMR 2 μg None DMG - 275 mM 3 Sucrose - 300 mM MSM - 275 mM

Example 2—Antigen Sparing Effect

Compositions A to F prepared in Example 1 were administeredsubcutaneously to BALB-c mice at day 0 and day 24. The protectiveeffects of the compositions against influenza were determined bymeasuring the haemmagglutination inhibition assay (HIA) titre of bloodtaken from the mice at various time points.

The haemmagglutination inhibition assay involved the following steps.

1. Sera was inactivated at 56° C. for 30 minutes.2. Non-specific chicken red blood cell (CRBC) agglutinating activity wasremoved by pre-incubation of sera with 1% CRBC for 30 minutes. CRBC wasremoved and the sera isolated.3. Sera was serially diluted in PBS/0.5% BSA.4. HAU of Influenza virus was added to wells.5. Plates were incubated for 30 minutes at room temperature.6. 1% CRBC/PBS suspension was added to wells.7. Plates were incubated for 30-45 minutes at room temperature.8. Agglutination in wells was determined by eye.9. HIA titre was recorded and reported, where HIA titre is thereciprocal of the greatest dilution series forming a CRBC pellet.

The results are depicted in FIG. 1. These results for Compositions D toF demonstrate that the combination of excipients with the heattreatment, resulted in an equivalent or better HIA titre compared withthe control Composition A. This is unexpected because Compositions D toF contained only 1% by weight of antigen compared to the control(compare with results obtained with Composition B).

Example 3—Acceleration of Onset of Immunity

Compositions G to N prepared in Example 1 were administeredsubcutaneously to BALB-c mice at day 0 and day 24. The protectiveeffects of the compositions against influenza were determined at varioustime points by measuring the HIA titre as described in Example 2.

The results are depicted in FIG. 2. The mice were considered to haveacquired immunity once the HIA titre was greater than 50. Immunity wasacquired most quickly with the combination of excipient, adjuvant andheat treatment (Composition N).

Example 4—Similar Results Observed with Different Excipients

Compositions O to R prepared in Example 1 were administeredsubcutaneously to BALB-c mice at day 0 and day 21. The protectiveeffects of the compositions against influenza were determined at varioustime points by measuring the HAI titre as described in Example 2.

The results are depicted in FIG. 3. These results demonstrate that theeffect on influenza antigen observed with polyethyleneimine in Examples1 and 2 are also observed with dimethylglycine. Thus, the antigensparing effect and acceleration of onset of immunity observed withpolyethyleneimine are also observed with dimethylglycine.

1. Use of an excipient which is a compound of formula (I) or aphysiologically acceptable salt or ester thereof:

wherein: R₁ represents hydrogen or C₁₋₆ alkyl; R₂ represents C₁₋₆ alkyl;and R₃ represents C₁₋₆ alkyl, for increasing the immunogenicity of aninfluenza antigen, which use comprises (a) freezing, (b) heat-treating,and/or (c) freeze-drying an aqueous composition comprising the influenzaantigen and the excipient.
 2. Use according to claim 1, in which thecompound of formula (I) is dimethylglycine or trimethylglycine.
 3. Useaccording to claim 2, in which the compound of formula (I) isdimethylglycine.
 4. Use according to any one of claims 1 to 3, in whichthe aqueous composition further comprises one or more sugars.
 5. Useaccording to claim 4, in which the one or more sugars are sucrose andraffinose.
 6. Use according to any one of the preceding claims, in whichthe aqueous composition further comprises an adjuvant.
 7. Use accordingto claim 6, in which the adjuvant is an aluminium salt adjuvant
 8. Useaccording to any one of the preceding, in which the immunogenicity ofthe influenza antigen is increased during the (a) freezing, (b)heat-treating, and/or (c) freeze-drying of the aqueous composition.
 9. Amethod for increasing the immunogenicity of an influenza antigen, saidmethod comprising (a) freezing, (b) heat-treating, and/or (c)freeze-drying an aqueous composition comprising the influenza antigen,an excipient as defined in any one of claims 1 to 3, optionally one ormore sugars as defined in claim 4 or 6 and optionally an adjuvant asdefined in claim 6 or
 7. 10. A vaccine composition obtainable by themethod defined in claim
 9. 11. A vaccine composition as defined in claim10, for use in the prevention of an influenza infection in a human oranimal patient.
 12. A vaccine composition for use according to claim 11,wherein the patient is (a) a child, an elderly patient and/or a patientsuffering from lung diseases, diabetes, cancer, or kidney or heartproblems, or (b) a patient who has previously suffered anadverse-reaction to an influenza vaccine.
 13. Use of a vaccinecomposition as defined in claim 10, in the manufacture of a medicamentfor preventing an influenza infection in a human or animal patient asdefined in claim 11 or
 12. 14. A method of preventing an influenzainfection in a human or animal patient as defined in claim 11 or 12, themethod comprising administering to said patient a vaccine composition asdefined in claim 10.